Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase

Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN+) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN+-based reducing power. This is based on an orthogonal, NMN+-dependent glycolytic pathway in Escherichia coli which can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN+. With a throughput of >106 variants per iteration, the growth selection discovers a Lactobacillus pentosus NADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that instead of directly contacting NCBs, the mutations optimize the enzyme’s global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme’s access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes.

Selection strain MX502 expressing either Lb NOX (NADH oxidase), TP NOX (NADPH oxidase), XenA WT (broad cofactor specificity), or XenA D116E (increased activity compared to WT and broad cofactor specificity) was plated on selection media supplemented with 0, or 2 mM NMN + and was incubated at 30° C for an extended growth period (14 days) to heighten contrast of growth between strains expressing NMNH active or inactive redox partners. Supplementation of NMN + in media can support minimal conversion of glucose to gluconate. Isolated colonies likely have extensive access to the NMN + present to enable growth, while clustered cells are highly limited and required active NMN(H) cycling to support continued growth. However, an active redox partner is essential to efficient and sustained glucose utilization. Although growth rate of single colonies was indicative of NMNH cycling activity, robust clustered growth after re-streaking onto fresh selection media was more predictive of true positives from selection. Figure 3. Growth rate of selection strains compared to wild-type E. coli strain. Growth of wild type (BW25113), MX 501, MX502 and MX503 harboring GDH Ortho and XenA was monitored in M9 minimal media with 20 g/L D-glucose and 0, 2, or 5 mM NMN + supplement. All strains possess the gene for initial glucose entry (a glucose facilitated diffusion porin from Zymomonas mobilis (Zm Glf). Selection strains MX503 (blue square) and MX502 (green diamond) show growth dependence on extracellular NMN + supplement while BW 25113 (grey circle) does not. Without heterologous expression of the NMN + producing gene (Francisella tularensis, NadEV) to help built up intracellular NMN + pool, MX 501 (orange triangle) is not able to grow, suggesting that in vivo NMN + biosynthesis still plays an essential role in selection strain growth, even when exogenous NMN + is supplied. Notably, while the selection strains exhibited sustained growth and approached similar final cell density to that of wild type, the growth rates were still slow. This may originate from the disruption of the heavily utilized EMP glycolysis, which cannot be fully replaced by the ED pathway, which naturally sustains a much lower carbon flux due to E. coli's regulatory control 1 . For MX501 with 0 or 2 mM NMN + supplement, MX503 with 0, 2, or 5 mM NMN + supplement, n = 2 biologically independent replicates. For all other samples, n = 3 biologically independent replicates. Data are presented as mean ± standard deviation. Source data are provided as a Source Data file. bound. D177 is found on the end of the second Rossman beta strand and is known to regulate specificity between NADPH and NADH. Due to its active role discriminating against NADPH, we expect that it may affect NMNH selectivity as well. G154 and G241 are on different loops that compose the adenine cleft, mutations at these positions would alter the volume of the binding pocket and create steric hindrance preventing the larger NADH from fitting, binding of the smaller NMNH is expected to be minimally affected. G156, Y157, I158, and G159 are found at the N-terminal region of the first Rossman alpha helix, these residues align the helix against the NADH pyrophosphate and create a set of polar backbone contacts and positive charge dipole to latch onto the cofactor. Substitutions in this region would reposition the helix to allow more favorable interactions with the NMNH phosphate. P295 is located across the NMNH ribose hydroxyls and mutation may create novel polar interaction. B, Specific enzymatic activities of the rational design mutants towards 0.3 mM NMNH. Compared to wild type, I158S and D177N exhibit ~2-fold or ~3-fold increased NMNH activity, respectively. While the mechanism of I158S can be readily rationalized as creating a novel hydrogen bond with the phosphate of NMNH, D177N may be playing a global and indirect role based on its far distance from NMNH. Therefore, D177 is determined as an ideal site for subsequent semi-rational engineering, where I158S is fixed. Compared to the data in Figure 2A, these data were generated using a slightly different method where 0.15mM FAD supplementation in protein purification was omitted.

Supplementary
Values represent the mean the two biological replicates. For statistics, WT to I158S, p= 0.0011; WT to D177N, p= 0.00041. The statistical significance was determined by two-tail t-tests (**: p < 0.005). Source data are provided as a Source Data file. increased NMNH-dependent activity. However, adding V240L to I158S did not further increase the activity significantly, and adding D177W to I158S resulted in very poor expression possibly due to impaired protein folding. Importantly, in LP 7 (I158S-D177W-V240L) where both V240L and D177W were combined with I158S, the activity increased ~2-fold compared to I158S, and the expression level was restored. These results are consistent with a non-additive, cooperative effect between V240L and D177W, which are located at the opposing sides of the adenosine binding cleft and predicted to pack against each other via hydrophobic interaction. Our models indicate that the dense packing mitigates Lp Nox's excessive flexibility when utilizing the much smaller non-canonical cofactors compared to NADH and promotes robust cofactor binding and catalysis. V240L alone does not afford the activity enhancing effect. The lone hydrophobic mutation D177W on the protein surface may cause a thermodynamic penalty associated with the extra energy needed for its solvation, resulting in poor folding 2,3 . This penalty is compensated by the addition of V240L which forms stabilizing interaction with D177W and makes it more deeply embedded 4,5

NAD(P) + Native
Native biological redox cofactor. Extensive knowledge in engineering for improved activity or redox cofactor specificity switches 6 .
Increased cost of biological sourcing 7 .

NMN + Biologically sourced noncanonicals
Truncation of NAD+ offers more structural similarity compared to other noncanonical redox cofactors. This similarity may improve ability to engineer proteins to utilize this cofactor effectively relative to fully synthetic cofactors [8][9][10] . Established biological synthesis routes enable implementation in vivo 9,11,12 .
Increased cost of biological sourcing 7 .

BNA + Synthetic noncanonicals
Facile chemical synthesis methods available leading to reduced input costs 13,14 .
High structural deviation requires more extensive engineering relative to NMN + 8 . Decreased stability relative to NAD(H) 15 .

MNA + Synthetic noncanonicals
Facile chemical synthesis methods available leading to reduced input costs 13,14 .
High structural deviation requires more extensive engineering relative to NMN + 8 . Decreased stability relative to NAD(H) 15 .